Immunoproteomic analysis of Lawsonia intracellularis identifies candidate neutralizing antibody targets for use in subunit vaccine development.

Lawsonia intracellularis is an obligate intracellular microorganism and the causative agent of porcine proliferative enteropathy. Due to its obligate intracellular nature, characterization of antigens and proteins involved in host-pathogen interaction and immune recognition have been difficult to achieve using conventional microbiological techniques. In this work, we used 2-dimensional gel electrophoresis coupled with Western-immunoblotting, mass spectrometry and bioinformatics to identify bacterial proteins that interact in vitro with pig intestinal cells (IPEC-1), have immunogenic properties and the potential to be used as subunit vaccine antigens. We detected eleven immunogenic bacterial proteins from which fliC (LI0710), LI1153 (annotated by NCBI as Putative protein N), and LI0649 (annotated as autotransporter) were predicted to be expressed on the outer membrane while LI0169 (oppA; annotated as ABC dipeptide transport system) was predicted to be periplasmic with a transmembrane domain forming a central pore through the plasma membrane. Genes coding for these four proteins were cloned and expressed in Escherichia coli and the corresponding recombinant proteins were purified using affinity chromatography. Porcine hyperimmune serum against whole Lawsonia lysate established that all four recombinant proteins were immunogenic. Further, rabbit hyperimmune sera generated against the vaccine strain of L. intracellularis and rabbit serum specific for each recombinant protein showed an inhibitory effect on the attachment and penetration of live, avirulent L. intracellularis, thus indicating that each protein is a potential neutralizing antibody target and a candidate for subunit vaccine formulation.